Regulated unfolding is critically important in the lifecycle of many proteins, such as those translocated across mitochondrial and chloroplast membranes, as well as those degraded by ATP-dependent proteases such as the proteasome. About half of all proteins synthesized in the eukaryotic cell are transported into or across a membrane. The protein translocation machineries are well defined biologically, but the means by which they transport and unfold proteins are not well understood at the biochemical and biophysical level. In contrast to protein folding, the mechanism of protein unfolding in the living cell has not been studied previously. The aims of this proposal are to understand the structural changes that occur in the unfolding protein prior to translocation and the molecular mechanisms of the unfolding machinery. The pathways of unfolding for a range of model proteins that translocate across membranes will be determined and compared with the pathway of spontaneous unfolding in solution. The mechanism of the unfoldase will be determined by inhibiting candidates either chemically or by mutation and measuring the effect on unfolding. The components of the import machinery that contribute to unfolding will be identified and the way in which they interact with each other the substrate protein determined. The hypothesis that the machinery unravels proteins by a physical pulling mechanism will be tested. This information is necessary to understand protein unfolding processes in the cell and to understand the function of a complex protein machine. The conclusions will also have broad implications for the understanding of protein translocation processes in the cell, in particular on the mechanisms that provide specificity to protein targeting to membranes. Interestingly, the unfolding processes during translocation and degradation share mechanistic features. Finally, the subject is directly relevant to human diseases. For example, an inherited form of oxalosis is due to the miss-sorting of an enzyme from peroxisomes to mitochondria. Since unfolding is not required for import into peroxisomes and the miss-sorted protein contains functional peroxisomal targeting information, preventing unfolding should correct the sorting defect.